System comprising elastase

ABSTRACT

The invention provides systems for treating an obstructed biological conduit that include administering to the conduit an agent that can degrade extracellular matrix of obstructing tissue. Particular methods include delivery of an enzyme or a mixture of several enzymes to the area or region of obstruction wherein the enzyme(s) have the capability to degrade extracellular matrix components within the obstruction thereby restoring the normal flow of transported fluid through the conduit. The invention also includes prophylactically dilating a section of conduit to minimize the risk of obstruction formation.

This application is a divisional of U.S. application Ser. No.11/454,554, filed Jun. 16, 2006, which is a divisional of U.S.application Ser. No. 09/669,051, filed Sep. 24, 2000 (U.S. Pat. No.7,063,838), which claims the benefit of U.S. Provisional ApplicationSer. No. 60/155,938, filed Sep. 24, 1999, each of which is incorporatedby reference herein in its entirety.

STATEMENT REGARDING GOVERNMENT RIGHTS

This invention was made with government support under grant no.HL07712-07 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of opening obstructedbiological conduits. Preferred methods of the invention include methodsand systems for opening obstructed biological conduits using localdelivery of a therapeutic agent, particularly a protease, to lyse theextracellular matrix of the obstructing tissue.

2. Background

Obstructions to biological conduits frequently result from trauma to theconduit which can result from transplant, graft or other surgicalprocedures wherein the extracellular matrix of the obstructing tissuelargely comprises collagen. Balloon angioplasty is a common initialtreatment for stenosis or stricture obstruction that yields excellentinitial results (Pauletto, Clinical Science, (1994) 87:467-79). However,this dilation method does not remove the obstructing tissue.

It only stretches open the lumen, the trauma of which has beenassociated with the release of several potent cytokines and growthfactors that can cause an injury which induces another round of cellproliferation, cell migration toward the lumen and synthesis of moreextracellular matrix. Consequently, balloon angioplasty is associatedwith restenosis in nearly all patients (Pauletto, Clinical Science,(1994) 87:467-79). There is currently no treatment that can sustainpatency over the long term.

The extracellular matrix, which holds a tissue together, is composedprimarily of collagen, the major fibrous component of animalextracellular connective tissue (Krane, J. Investigative Dermatology(1982) 79:83s-86s; Shingleton, Biochem. Cell Biol., (1996) 74:759-75).The collagen molecule has a base unit of three stands of repeating aminoacids coiled into a triple helix. These triple helix coils are thenwoven into a right-handed cable. As the collagen matures, cross-linksform between the chains and the collagen becomes progressively moreinsoluble and resistant to lysis. When properly formed, collagen has agreater tensile strength than steel. Not surprisingly, when the bodybuilds new tissue collagen provides the extracellular structuralframework such that the deposition of hard collagen in the lesion canresult in duct obstruction.

Benign biliary stricture results in obstruction of the flow of bile fromthe liver and can result in jaundice and hepatic dysfunction. Ifuntreated, biliary obstruction can result in hepatic failure and death.Biliary strictures can form after duct injury during cholecystectomy.They can also from at biliary anastomoses after liver transplantationand other biliary reconstructive surgeries (Vitale, Am. J. Surgery(1996) 171:553-7; Lilliemoe, Annals of Surgery (1997) 225).

Historically, benign biliary stricture has been treated surgically byremoving the diseased duct segment and reconnecting the duct end-to-end,or connecting the duct to the bowel via a hepaticojejunostomy loop(Lilliemoe, Annals of Surgery (1997) 225). These long and difficultsurgeries have significant morbidity and mortality due to bleeding,infection, biliary leak, and recurrent biliary obstruction at theanastomosis. Post-operative recovery takes weeks to months. Morerecently, minimally invasive treatments such as percutaneous balloondilation have been utilized, yielding good initial biliary patencyresults (Vitale, Am. J. Surgery (1996) 171:553-7, Lilliemoe, Annals ofSurgery (1997) 2250). However, balloon dilation causes a localizedinjury, inducing a healing response that often results in restenosis(Pauletto, Clinical Science, (1994) 87:467-79). Long-term stenting atthe common bile duct with flexible biliary drainage catheters is anotherminimally invasive alternative to surgery (Vitale, Am. J. Surgery (1996)171:553-7). However, these indwelling biliary drainage catheters oftenbecome infected, or clogged with debris, and must be changed frequently.At present, long-term treatment of biliary stricture remains a difficultclinical problem.

Patients with chronic, end-stage renal failure may require replacementof their kidney function in order to survive. In the United States,long-term hemodialysis is the most common treatment method for end stagechronic renal failure. In 1993, more than 130,000 patients underwentlong term hemodialysis (Gaylord, J. Vascular and InterventionalRadiology (1993) 4:103-7); more than 80% of these patients implementhemodialysis through the use of a synthetic arteriovenous graft (Windus,Am. J. Kidney Diseases (1993) 21:457-71). In a majority of thesepatients, the graft consists of a 6 mm Gore-Tex tube that is surgicallyimplanted between an artery and a vein, usually in the forearm or upperarm. This high flow conduit can then be accessed with needles forhemodialysis sessions.

Nearly all hemodialysis grafts fail, usually within two years, and a newgraft must be created surgically to maintain hemodialysis. Thesepatients face repeated interruption of hemodialysis, and multiplehospitalizations for radiological and surgical procedures. Since eachsurgical graft revision consumes more available vein, eventually theyare at risk for mortality from lack of sites for hemodialysis access.One estimate placed the cost of graft placement, hemodialysis, treatmentof complications, placement of venous catheters, hospitalization costs,and time away from work at as much as $500 million, in 1990 alone(Windus, Am. J. Kidney Diseases (1993) 21:457-71).

The most frequent cause of hemodialysis graft failure is thrombosis,which is often due to development of a stenosis in the vein justdownstream from the graft-vein anastomosis (Safa, Radiology (1996)199:653-7. Histologic analysis of the stenosis reveals a firm, pale,relatively homogeneous lesion interposed between the intimal and mediallayers of the vein which thickens the vessel wall and narrows the lumen(Swedberg, Circulation (1989) 80:1726-36). This lesion, which has beengiven the name intimal hyperplasia is composed of vascular smooth musclecells surrounded by an extensive extracellular collagen matrix(Swedberg, Circulation (1989) 80:1726-36; Trerotola, J. Vascular andInterventional Radiology (1995) 6:387-96). Balloon angioplasty is themost common initial treatment for stenosis of hemodialysis grafts andyields excellent initial patency results (Safa, Radiology (1996)199:653-7). However, this purely mechanical method of stretching openthe stenosis causes an injury which induces another round of cellproliferation, cell migration toward the lumen and synthesis of moreextracellular matrix. Consequently, balloon angioplasty is associatedwith restenosis in nearly all patients (Safa, Radiology (1996)199:653-7). There is currently no treatment which can sustain thepatency of synthetic arteriovenous hemodialysis grafts over the longterm.

Intimal hyperplasia research has focused largely on the cellularcomponent of the lesion. The use of radiation and pharmaceutical agentsto inhibit cell proliferation and migration are active areas of research(Hirai, ACTA Radiologica (1996) 37:229-33; Reimers, J. InvasiveCardiology (1998) 10:323-31; Choi, J. Vascular Surgery (1994)19:125-34). To date, the results of these studies have been equivocal,and none of these new treatments has gained wide clinical acceptance.This matrix is composed predominantly of collagen and previous work inanimals has demonstrated that systemic inhibition of collagen synthesisdecreases the production of intimal hyperplasia (Choi, Archives ofSurgery (1995) 130:257-261).

During normal tissue growth and remodeling, existing collagen matricesmust be removed or modified. This collagen remodeling is carried out bymacrophages and fibroblasts, two cell types which secrete a distinctclass of proteases called “collagenases” (Swedberg, Circulation (1989)80:1726-36; Trerotola, J. Vascular and Interventional Radiology (1995)6:387-96; Hirai, ACTA Radiologica (1996) 37:229-33). These collagenasesrapidly degrade insoluble collagen fibrils to small, soluble peptidefragments, which are carried away from the site by the flow of blood andlymph.

See also U.S. Pat. Nos. 5,981,568; 5,409,926; and 6,074,659.

It thus would be desirable to provide new methods to relieveobstructions blocking flow through biological conduits.

SUMMARY OF THE INVENTION

I have now found new methods and systems for relieving an obstruction ina biological conduit, e.g. mammalian vasculature. Methods of theinvention include administration to an obstruction site of a therapeuticagent that can preferably degrade (in vivo) the extracellular matrix ofthe obstructing tissue, particularly collagen and/or elastin. Preferredmethods of the invention include administration to an obstruction of anenzyme or a mixture of enzymes that are capable of degrading keyextracellular matrix components (including collagen and/or elastin)resulting in the solubilization or other removal of the obstructingtissue.

Methods and systems of the invention can be applied to a variety ofspecific therapies. For example, methods of the invention includetreatment of biliary stricture with the use of exogenous collagenase,elastase or other agent, whereby an enzyme composition comprisingcollagenase, elastase or other agent is directly administered to or into(such as by catheter injection) the wall of the lesion or otherobstruction. The enzyme(s) dissolves the collagen and/or elastin in theextracellular matrix, resulting in the solubilization of fibrous tissuefrom the duct wall near the lumen, and a return of duct flow or opening.

Methods of the invention also include pretreating an obstruction (e.g.in a mammalian duct) with collagenase, elastase or other agent tofacilitate dilation such that if treatment under enzymatic degradationconditions alone is insufficient to reopen a conduit, then conventionaltreatment with e.g. balloon dilation is still an option. It has beenfound that enzymatic degradation pre-treatment in accordance with theinvention can improve the outcome of balloon dilation since enzymetreatment partially digests the collagen fibrils. Therefore, the overalleffect will be a softening of the remaining tissue. The softened tissueis more amenable to balloon dilation at lower pressures, resulting inless mechanical trauma to the duct during dilation.

Preferably, the therapeutic agent is delivered proximately to a targetedsite, e.g. by injection, catheter delivery or the like.

A variety of therapeutic agents may be employed in the methods of theinvention. Suitable therapeutic agents for use in the methods andsystems of the invention can be readily identified, e.g. simply bytesting a candidate agent to determine if it reduces an undesiredvasculature obstruction in a mammal, particularly a coronary obstructionin a mammalian heart. Preferred therapeutic agents comprise one or morepeptide bonds (i.e a peptidic agent), and typically contain at least 2,3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids, preferably one or moreof the natural amino acids. Preferred therapeutic agents include largemolecules, e.g. compounds having a molecular weight of at least about1,000, 2,000, 5,000 or 10,000 kD, or even at least about 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000 kD.

Specifically preferred therapeutic agents for use in the methods andsystems of the invention include proteases and other enzymes e.g. acollagenase such as Clostridial collagenase, a proteolytic enzyme thatdissolves collagen, and/or an elastase such as a pancreatic elastase, aproteolytic enzyme that dissolves elastin. Preferred delivery ofcollagenase and other therapeutic agents of the invention includedirectly injecting the agent into the target lesion or otherobstruction. Preferably, a homogeneous distribution of a therapeuticenzyme or enzyme mixture is administered to a target site with a drugdelivery catheter. The therapeutic agent can then dissolve the keyextracellular collagen components necessary to solubilize theobstructing tissue from the vessel wall near the lumen.

Treatment methods of the invention provide significant advantages overprior treatment methodologies. For example, enzymatic degradation of oneor more key components of the extracellular matrix gently removes thetissue obstructing the lumen. Additionally, collagenolysis or othertherapeutic administration is relatively atraumatic. Moreover,collagenase also can liberate intact, viable cells from tissue.Therefore, treatment methods of the invention can remove both the sourceof mechanical obstruction and a source of cytokines and growth factors,which stimulate restenosis.

A single or combination of more than one distinct therapeutic agents maybe administered in a particular therapeutic application. In this regard,a particular treatment protocol can be optimized by selection of anoptimal therapeutic agent, or optimal “cocktail” of multiple therapeuticagents. Such optimal agent(s) for a specific treatment method can bereadily identified by routine procedures, e.g. testing selectedtherapeutic agents and combinations thereof in in vivo or in vitroassays.

In another aspect of the invention, treatment compositions and treatmentkits are provided. More particularly, treatment compositions of theinvention preferably contain one or more enzymatic agents such ascollagenase preferably admixed with a pharmaceutically acceptablecarrier. Such compositions can be suitable packaged in conjunction withan appropriate delivery tool such as an injection syringe or a deliverycatheter. The delivery device and/or treatment solution are preferablypackaged in sterile condition. The delivery device and treatmentcomposition can be packaged separately or in combination, more typicallyin combination. The delivery device preferably is adapted for in situ,preferably localized, delivery of the therapeutic agent directly intothe targeted biological conduit obstruction.

Typical subjects for treatment in accordance with the invention includemammals, particularly primates, especially humans. Other subjects may betreated in accordance with the invention such as domesticated animals,e.g. pets such as dogs, cats and the like, and horses and livestockanimals such as cattle, pigs, sheep and the like. Subjects that may betreated in accordance with the invention include those mammals sufferingfrom or susceptible to biliary stricture including benign biliarystricture, stenosis of hemodialysis graft, intimal hyperplasia, and/orcoronary obstruction, and the like. As discussed above, methods of theinvention may be administered as a pre-treatment protocol before anothertherapeutic regime such as a balloon angioplasty; during the course ofanother therapeutic regime, e.g. where a therapeutic composition of theinvention is administered during the course of an angioplasty or otherprocedure; or after another treatment regime, e.g. where a therapeuticcomposition of the invention is administered after an angioplasty oradministration of other therapeutic agents.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a common bile duct in a dog with a high grade stricture;

FIG. 2 shows a common bile duct in a dog with a high grade strictureafter treatment;

FIG. 3 is a histology picture of a normal common bile duct from a dog;

FIG. 4 is a histology picture of a common bile duct stricture from a dogwith a high grade stricture before treatment;

FIG. 5 is a histology picture of a common bile duct stricture from a dogafter treatment with collagenase wherein the arrows denote the outerlimit of collagen breakdown; and

FIG. 6 shows a normal common bile duct in a dog.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of introducing a therapeuticagent that is capable of degrading an extracellular matrix component tothereby facilitate the reopening of a constricted biological conduit. Inparticular, the invention provides for introduction to an obstructedbiological conduit of a therapeutic agent that degrades collagen and/orelastin. The present invention further provides methods of dilating abiological conduit by introducing a therapeutic agent into a biologicalconduit, preferably an isolated segment of the conduit.

In one embodiment of the present invention, the degradation of astricture, lesion or other obstruction is accomplished by introducingone or more therapeutic agents that are capable of degrading one or moreextracellular matrix components thereby facilitating the reopening ofthe constricted segment of the conduit. Major structural components ofthe extracellular matrix include collagen and elastin.

Preferred therapeutic agents for use in accordance with the inventionare able to interact with and degrade either one or both of collagen andelastin.

As discussed above, a variety of compositions may be used in the methodsand systems of the invention. Preferred therapeutic compositionscomprise one or more agents that can solubilize or otherwise degradecollagen or elastin in vivo. Suitable therapeutic agents can be readilyidentified by simple testing, e.g. in vitro testing of a candidatetherapeutic compound relative to a control for the ability to solubilizeor otherwise degrade collagen or elastin, e.g. at least 10% more than acontrol.

More particularly, a candidate therapeutic compound can be identified inthe following in vitro assay that includes steps 1) and 2):

1) contacting comparable mammalian tissue samples with i) a candidatetherapeutic agent and ii) a control (i.e. vehicle carrier without addedcandidate agent), suitably with a 0.1 mg of the candidate agentcontacted to 0.5 ml of the tissue sample; and

2) detecting digestion of the tissue sample by the candidate agentrelative to the control. Digestion can be suitably assessed e.g. bymicroscopic analysis. Tissue digestion is suitably carried out in awater bath at 37° C. Fresh pig tendon is suitably employed as a tissuesample. The tissue sample can be excised, trimmed, washed blotted dryand weighed, and individual tendon pieces suspended in 3.58 mg/ml HEPESbuffer at neutral pH. See Example 1 which follows for a detaileddiscussion of this protocol. Such an in vitro protocol that containssteps 1) and 2) is referred to herein as a “standard in vitro tissuedigestion assay” or other similar phrase.

Preferred therapeutic agents for use in accordance with the inventioninclude those that exhibit digestion activity in such a standard invitro tissue digestion assay at least about 10 percent greater relativeto a control, more preferably at least about 20% greater digestionactivity relative to a control; still more preferably at least about30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater digestion activityrelative to a control.

Appropriate therapeutic agents can comprise at least one and frequentlyseveral enzymes such that the therapeutic agent is capable of degradingboth significant matrix components of tissue obstruction. Particularlypreferable therapeutic agents will comprise either a collagenase orelastase or both. Specifically preferred are therapeutic agentscomprising a highly purified, injectable collagenase preparation such asthat produced from cultures of Clostridium histolyticum by BioSpecificsTechnologies Corporation (Lynbrook, N.Y.). This enzyme preparation iscomposed of two similar but distinct collagenases. The Clostridialcollagenases cleave all forms of collagen at multiple sites along thehelix, rapidly converting insoluble collagen fibrils to small, solublepeptides. Also preferable are therapeutic agents comprising elastase,particularly pancreatic elastase, an enzyme capable of degradingelastin. Trypsin inhibitors also can be suitably employed as thetherapeutic agent in the methods of the invention.

In a further aspect of the present invention, the methods furtherinclude means to prevent damage to tissue that is not associated withconduit obstruction. Preferred enzymes incorporated in the therapeuticagents are large (>100,000 kD) and diffuse slowly in the extracellularcompartment after injection. Further, collagenases comprise a domain (inaddition to the active site) which binds tightly to tissue.Consequently, these enzymes remain largely contained withincollagen-rich target tissues after injection. Also, the enzyme'sactivity is quickly extinguished in the blood pool by circulatinginhibitors. Therefore, injected collagenase, which diffuses from theinterstitial compartment into the blood pool, will be rapidly inhibited,preventing systemic side effects.

Fragments of therapeutic agents also can be administered to a patient inaccordance with the invention. For example, fragments of theabove-mentioned collagenases and elastases can be administered to apatient provided such fragments provide the desired therapeutic effect,i.e. degradation of obstruction of a biological conduit. As referred toherein, a collagenase, elastase or other enzyme includes therapeuticallyeffective fragments of such enzymes.

In certain preferred aspects of the invention, the therapeutic agent(s)that are administered to a patient are other than a cytostatic agent;cytoskeletal inhibitor; an aminoquinazolinone, particularly a6-aminoquinazolinone; a vascular smooth muscle protein such asantibodies, growth hormones or cytokines.

In specific embodiments, the degradation of elastin, an extracellularmatrix component that contributes to tissue elasticity, is notdesirable. Therapeutic agents comprising only enzymes, which do notdegrade elastin, such as collagenases, can be employed. Therefore, theelastic properties of the conduit wall will likely be preserved aftertreatment.

In a preferred aspect of the invention, a therapeutic agent comprisingat least one enzyme capable of degrading elastin, collagen or both isdelivered to the targeted obstruction site with a catheter. Preferredcatheters are capable of directly localizing a therapeutic agentdirectly into the extracellular matrix of the obstruction. Particularlypreferable catheters are capable of delivering accurate doses of thetherapeutic agent with an even distribution over the entire obstructedarea of the conduct. One particularly preferred example of a catheterfor use in the method of the present invention is the Infiltrator®catheter produced by InterVentional Technologies Corporation (IVT) (SanDiego, Calif.), which delivers a precisely controlled dosage of a drugdirectly into a selected segment of vessel wall (FIG. 1) (Reimers, J.Invasive Cardiology (1998) 10:323-331; Barath, Catheterization andCardiovascular Diagnosis (1997) 41:333-41; Woessner, Biochem. Cell Biol.(1996) 74: 777-84). Using this preferred catheter a therapeutic agentcan be delivered at low pressure via a series of miniaturized injectorports mounted on the balloon surface. When the positioning balloon isinflated, the injector ports extend and enter the vessel wall over the3600 surface of a 15 mm segment of vessel. Each injector port is lessthan 0.0035 inch in size. Drug delivery can be performed in less than 10seconds, with microliter precision and minimal immediate drug washout.The injected drug is delivered homogeneously in the wall of the vesselor duct (FIG. 2). The triple lumen design provides independent channelsfor guidewire advancement, balloon inflation and drug delivery. Traumaassociated with injector port penetration is minimal and the long-termhistologic effects are negligible (Woessner, Biochem. Cell Biol. (1996)74: 777-84). In addition, the device has been engineered such that theinjector ports are recessed while maneuvering in the vessel.Additionally, the Infiltrator® catheter is capable of balloon inflationwith sufficient force for angioplasty applications. The excellentcontrol of drug delivery observed with Infiltrator® can be significantsince preferred therapeutic agents of the present invention potentiallycan degrade collagen and/or elastin in nearly all forms of tissue in anon-specific manner.

In yet another embodiment of the present invention, a therapeutic doseis employed which will restore conduit flow while maintaining conduitwall integrity. Several parameters need to be defined to maximize methodefficiency, including the amount of enzyme to be delivered, and thevolume of enzyme solution to be injected so that the reopening of theconduit occurs with a single dose protocol. Ideally repeat or multipledosing is reserved only for patients who have an incomplete response tothe initial injection.

In regards to the volume of therapeutic agent solution delivered,preferably the conduit wall is not saturated completely, as this canlead to transmural digestion and conduit rupture. Instead, the optimaldose is determined by targeting the thickness of the wall (from theoutside in) which needs to be removed in order to restore adequate flow,while leaving the remaining wall intact. An overly dilute solution willbe ineffective at collagen lysis while an overly concentrated solutionwill have a higher diffusion gradient into the surrounding tissues,thereby increasing the risk of transmural digestion and rupture.

Collagenase doses are generally expressed as “units” of activity,instead of mass units. Individual lots of collagenase are evaluated forenzymatic activity using standardized assays and a specific activity(expressed in units/mg) of the lot is determined. BTC uses an assay thatgenerates “ABC units” of activity. The specific activity of othercollagenase preparations are sometimes expressed in the older “Mandelunits”. One ABC unit is roughly equivalent to two Mandel units.

Preferable doses and concentrations of enzyme solution are between 1000and 20000 ABC units, more preferable are between 2500 and 10000 ABCunits and enzyme doses of 5,000 ABC units in 0.5 ml of buffer are mostpreferred.

It will be appreciated that actual preferred dosage amounts of othertherapeutic agents in a given therapy will vary according to e.g. thespecific compound being utilized, the particular composition formulated,the mode of administration and characteristics of the subject, e.g. thespecies, sex, weight, general health and age of the subject. Optimaladministration doses for a given protocol of administration can bereadily ascertained by those skilled in the art using dosagedetermination tests, including those described above and in the exampleswhich follow.

Therapeutic agents of the invention are suitably administered as apharmaceutical composition with one or more suitable carriers.Therapeutic agents of the invention are typically formulated ininjectable form, e.g. with the therapeutic agent dissolved in a suitablefluid carrier. See the examples which follow for preferred compositions.

As discussed above, the methods and systems of the invention can beemployed to treat (including prophylactic treatment) a variety ofdiseases and disorders. In particular, methods and systems of theinvention can be employed to relieve or otherwise treat a variety oflesions and other obstructions found in common bile ducts or vascularsystems. Methods of the invention are also useful to relieve lesions andother obstructions in other biological conduits including e.g. ureterer,pancreatic duct, bronchi, coronary and the like.

The invention also includes prophylactic-type treatment, e.g. methods todilate a biological conduit whereby the increased conduit diameterobviates the potential of obstruction formation with a conduit.Temporary and partial degradation of the elastin component of a conduitwall reduces the elasticity of the conduit, thereby facilitatingmodifications of the size and shape of the conduit. Introducing a doseof therapeutic agent in accordance with the invention into the lumen ofan isolated conduit or some section thereof results in complete orpartial diffusion of the therapeutic agent into the wall of the isolatedconduit during a specified period of time. Subsequent pressurization ofthe treated region, either while the region is still isolated or afterremoving the means of isolation, increases the lumen diameter bydilation. Regeneration of the conduit elastin framework results in aconduit with a larger lumen diameter without compromising the structuralintegrity.

Arteriovenous hemodialysis grafts are frequently placed in the arm ofthe patient such that blood can be withdrawn and purified blood returnedthrough the graft. Frequently the lumenal diameter of the venous outflowis smaller than the graft lumenal diameter. Development of a stenosisdue to intimal hyperplasia can further reduce the lumenal diameter ofthe venous outflow such that an insufficient volume of blood passesthrough the venous outflow. To prevent intimal hyperplasia and stenosisformation, dilating the venous outflow vein using the above describedmethod of partially degrading the elastin component of the vascular walldownstream of the site of graft implantation such that the lumenaldiameter of the venous outflow is similar to or larger than the diameterof the interposed loop graft reduces the likelihood of forming astenosis due to intimal hyperplasia. Venous dilation can be performedeither before or after interposing a graft between the artery and vein.

All documents mentioned herein are incorporated herein by reference. Thepresent invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Tissue Digestion Analysis

The protocol of the following example is a detailed description of a“standard in vitro tissue digestion assay” as referred to herein.

The rate of tissue digestion, which is composed mostly of collagen, by amixture of collagenase and elastase, proteolytic enzymes with activityrespectively against collagen and elastin, was determined. Trypsininhibitor was added to negate the effect of any residual trypsinactivity. Briefly, fresh pig tendon was excised, trimmed, washed,blotted dry and weighed. Individual tendon pieces were suspended in 3.58mg/ml HEPES buffer at neutral pH and various concentrations of enzymeswere added. Iodinated radiographic contrast was added in variousconcentrations to some of the enzyme solutions. The tissue digestion wascarried out in a water bath at 37° C. At various time points, the tendonpieces were removed from the enzyme solution, washed, blotted dry andweighed. Each time point was derived from the average of three samples.The effect of enzyme concentration on tissue digestion rates wasstudied. As expected, increasing the concentration of enzymes in vitroincreased the rate of tissue digestion (FIG. 3). Buffer alone had noeffect on the tissue. Extrapolating digestion rates in vitro to an invivo situation has proven difficult. For Dupuytren's contractures, theeffective dose for transecting fibrous cords in vitro was 500 ABC units.However, the effective in vivo dose was 10,000 ABC units.

The effect of iodinated radiographic contrast material on tissuedigestion rates was also studied (FIG. 4). This study was performed tomonitor enzyme delivery by mixing it with contrast prior to injection.These results demonstrate that Omnipaque 350 iodinated contrast materialinhibits enzyme activity at radiographically visible (35%)concentrations, but not at lower (1-5%) concentrations (FIG. 4). Similarresults were observed with Hypaque 60 contrast.

EXAMPLE 2 Determining Dose Dependent in Vitro Activity of a TherapeuticAgent Including Collagenase, Elastase, and a Trypsin Inhibitor

The effect of enzyme concentration on tissue digestion rates was studied(FIG. 3). The “1×” tissue sample was treated with collagenase 156 Mandelunits/ml+elastase 0.125 mg/ml+trypsin inhibitor 0.38 mg/ml. The “2×”sample was treated with collagenase 312 Mandel units/ml+elastase 0.25mg/ml+trypsin inhibitor 0.76 mg/ml. The “5×” sample was treated withcollagenase 780 Mandel units/ml+elastase 0.625 mg/ml+trypsin inhibitor1.9 mg/ml. All digestion volumes were 0.5 ml. Increasing theconcentration of enzymes in vitro increased the rate of tissue digestion(FIG. 3). Buffer alone had no effect on the tissue. An effective in vivodose was found to be 10,000 ABC units.

EXAMPLE 3 Determining the Effect of Iodinated Radiographic ContrastMaterial on Tissue Digestion Rates to Facilitate Monitoring EnzymeDelivery Prior to Injection of a Therapeutic Agent Comprising a ContrastMaterial into a Patient

The “35% Omnipaque” tissue sample was treated with collagenase 156Mandel units/ml+elastase 0.125 mg/ml+0.38 trypsin inhibitor with 35%Omnipaque 350 contrast (volume:volume). The “5% Omnipaque” sample wastreated with collagenase 312 Mandel units/ml+elastase 0.25 mg/ml+0.76trypsin inhibitor with 5% Omnipaque 350 (volume:volume). The “1%Omnipaque” sample was treated with collagenase 312 Mandelunits/ml+elastase 0.25 mg/ml+0.76 trypsin inhibitor with 1% Omnipaque350. All digestion volumes were 0.5 ml. These results demonstrate thatOmnipaque 350 iodinated contrast material inhibits enzyme activity atradiographically visible (35%) concentrations, but not at lower (1-5%)concentrations (FIG. 4). Similar results were observed with Hypaque 60contrast.

EXAMPLE 4 Creating a Stricture in the Common Bile Duct of Dogs andTreatment of the Resulting Stricture with Transcatheter IntramuralCollagenase Therapy

Right subcostal laparotomy was performed in dogs to expose thegallbladder, which was then affixed to the anterior abdominal wall of 11dogs (n=11). After 2 weeks, a single focal thermal injury was made inthe common bile duct (CBD) using a catheter with an electrocoagulationtip placed through the gallbladder access. A 4.8 Fr biliary stent wasplaced to prevent complete duct occlusion in 7 animals. Stricturedevelopment was monitored with percutaneous cholangiography over fiveweeks. Collagenase was then directly infused into the wall of thestrictured CBD using an Infiltrator drug delivery catheter (n=3). TheInfiltrator has three arrays of microinjector needles mounted on aballoon which extend and enter the duct wall over the 360-degreesurface. After treatment, internal plastic stents were placed in 2animals. Explants of the CBD were obtained the following day. H&E,trichrome, and elastin staining were used for histopathologic analysis.

CBD strictures were successfully created in 7/11 animals as determinedby cholangiography (FIG. 1). Failures were due to gallbladder leak (n=2)and perforation at the site of thermal injury (n=2). Histologic analysisof an untreated stricture demonstrated a thickened wall with acircumferential network of collagen bundles and associated lumenalnarrowing (FIG. 4). Strictures treated with collagenase demonstrated acircumferential lysis of collagen at the treatment site, with sparing ofthe normal duct, arteries and veins (FIGS. 2 and 5). All three animalsdeveloped bile leaks after treatment, two from the gallbladder accesssite and one from the treatment site. There was vascular congestion andinflammation in portions of the small bowel mucosa and peritoneum aftertreatment in all animals, to varying degrees.

EXAMPLE 5 Relief of Strictures in the Common Bile Duct of a Patient

A large dog was used as the patient such that under general anesthesia acholecystostomy tract was created and the gallbladder was “tacked” tothe abdominal wall with retention sutures. A cholangiogram was performedwith Hypaque-60, using a marker catheter, in order to define theanatomy. Then, a flexible catheter with a bipolar electrode tip wasconstructed as previously described (Becker, Radiology (1988) 167:63-8).This catheter was inserted through the gallbladder (FIG. 5) andpositioned with its “hot” tip (arrow) in the distal common bile ductsuch that the catheter was pulled back and the treatment was repeateduntil a 1.0 cm length of duct was injured (FIG. 6). Immediately afterdelivering the current there was a mild-moderate amount of smoothnarrowing of the treated segment of duct (arrow), possibly due to spasmor edema. A pigtail nephrostomy drainage catheter was then insertedthrough the fresh cholecystostomy tract into the gallbladder. The distalend was closed with an IV cap and buried in the subcutaneous tissue. Thesurgical wounds were then closed in a two-layer fashion.

After 7 days, a follow-up cholangiogram was performed to evaluate thethermally induced stenosis. A 20 gauge needle was used to percutaneouslyaccess the drainage catheter through the IV cap. A cholangiogram wasperformed demonstrating moderate-marked dilation of the biliary tree(FIG. 1). There was a high-grade stricture of the mid common bile duct,where the thermal injury had been made.

Strictures are created in five large dogs using the methods describedabove and in Example 4. In addition, an objective measurement of biliarypatency (the Whitaker study) is made of the common bile duct, bothbefore and after making a stricture. The Whitaker study is performed byinjecting normal saline through a catheter positioned in the common bileduct. Flow rates are increased and pressure measurements are taken untila peak pressure of 40 mmHg is reached.

The thermal lesions mature into fibrous strictures over a six weekperiod. One animal is then sacrificed and a histologic assessment ismade of the extrahepatic biliary tree. Samples are taken of the ductproximal to the lesion, the mid portion of the lesion (FIG. 4), thelesion edge, and the duct distal to the lesion. Assessments of 1) ductmorphology. 2) cell type and number, 3) the extent and appearance of theextracellular matrix, and 4) extent of epithelialization are made. Asecond animal is sacrificed after an additional 6 weeks after thermalinjury and a similar analysis carried out.

A cholangiogram is performed to visually assess the stricture (FIG. 1)and a Whitaker test is also performed on the remaining 3 dogs. Then, theInfiltrator catheter is then deployed within the lesion and 0.5 mL ofcollagenase preparation (10,000 Units/ml) is injected into the wall ofthe lesion. On post-treatment day 1, a follow-up cholangiogram andWhitaker test are performed.

In cases where incomplete response is noted, a second treatment can begiven and a second follow-up cholangiogram and Whitaker test isperformed the following day. Hepatic enzyme levels will be drawn toassess the effect of stricture and then treatment on hepatic function.Alternatively, incomplete response from collagenase can be followed upwith subsequent angioplasty or a combined collagenase/angioplastytreatment.

After treatment with collagenase, a final cholangiogram is taken after 1week (FIG. 2). At this time, the animal is sacrificed and theextrahepatic biliary tree harvested. Histologic assessments are made ofthe bile duct proximal to the treated lesion, the mid portion of thetreated lesion (FIG. 5), the treated lesion edge, and the duct distal tothe lesion. Assessments of 1) duct morphology, 2) cell type and number,3) the extent and appearance of the extracellular matrix, and 4) extentof epithelialization were made. FIG. 5 is a histology image of a commonbile duct stricture after treatment. The arrows denote the outer limitof collagen breakdown. The histological examination of the treatedcommon bile duct stricture demonstrates a circumferential lysis ofcollagen at the treatment site, while sparing damage to the normal duct,arteries and veins.

EXAMPLE 6 Relief of Stenosis Due to Intimal Hyperplasia of a SyntheticHemodialysis Graft

Standard, untapered 5 mm diameter polytetrafluoroethylene (PFTE) loopgrafts were interposed between the femoral artery and the femoral veinin the hind limbs of 25-35 kg dogs, as described previously (Trerotola,J. Vascular and Interventional Radiology (1995) 6:387-96). An end-to-endconfiguration had been selected to facilitate optimal positioning of thecatheter drug delivery balloon during treatment of a stenosis. Standard,cut-film angiography is performed one week after surgery to assess thearterial inflow, the artery-graft anastomosis, the vein-graftanastomosis, and the venous outflow. After this, routine physicalexamination of the grafts will be carried out to screen for patency.Twenty weeks after surgery, standard, cut-film angiography is performedto assess the lumenal diameter of the grafts and their venous outflow.At this time, a stenosis due to intimal hyperplasia is seen in thevenous outflow with an associated pressure gradient (Trerotola, J.Vascular and Interventional Radiology (1995) 6:387-96). Then, using thefirst animal, the therapy delivery catheter is deployed within a graftand 5000 ABC units of collagenase in 0.5 ml is infiltrated into the wallof the lesion at the venous outflow. The catheter is flushed and thecontralateral lesion receives 1 ml of saline, delivered in an identicalmanner. Nearly all collagenase activity is extinguished after 1-2 dayssuch that the grafts are re-examined with angiography after 3 days.Repeat measurements of lumenal diameter and invasive pressuremeasurements across the lesion are also taken. The animals aresacrificed and the grafts excised, pressure-fixed, and examinedhistologically. Assessments are made of the distal graft, the venousanastomosis, the mid-portion of the treated lesion, the lesion edge, andthe normal vein downstream from the graft. Additional assessments of 1)cell type, morphology and number, 2) extent of extracellular matrix, 3)overall adventitial, medial, and intimal thickness, 4) extent of intimalhyperplasia, and 5) extent of endothelialization are made.

EXAMPLE 7

Four dogs are used for a controlled study of collagenase treatment.Bilateral grafts are created as described previously and standard,cut-film angiography is performed one week after surgery to access thearterial inflow, the artery-graft anastomosis, the vein-graftanastomosis, and the venous outflow. After this, routine physicalexamination of the grafts is carried out to screen for patency. Then,twenty weeks after surgery, standard, cut-film angiography is performedto assess the lumenal diameter of the grafts and their venous outflow.An obvious stenosis due to intimal hyperplasia is usually seen in thevenous outflow with an associated pressure gradient (Trerotola, J.Vascular and Interventional Radiology (1995) 6:387-96). The Infiltratorcatheter is then deployed within the lesion and the selected dose ofcollagenase is infiltrated into the wall of the lesion. Thecontralateral, control graft is treated in an identical manner, exceptthat saline is delivered instead of collagenase. Three days aftertreatment, the grafts are restudied with angiography and invasivepressure measurements to determine the acute effects of collagenasetreatment. Changes in lumenal diameter and pressure gradients arecalculated for both the collagenase-treated group and the saline-treatedgroup and ten days after collagenase treatment, the grafts are studied afinal time. The animals are sacrificed and the grafts are excised,pressure-fixed, and examined histologically, as described above.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention as set forth in the following claims.

What is claimed is:
 1. A system for enlarging the diameter of an arteryin a human subject, said artery having a lumen, the system comprising:(a) a catheter configured for injecting a pharmaceutical compositiondirectly into a selected segment of the wall of the artery; and (b) apharmaceutical composition suitable for administration to the humansubject comprising an elastase, wherein said elastase is present in adose sufficient to enlarge the diameter of the artery and the diameterof the lumen of the artery when administered via the catheter to thewall of the artery in the human subject.
 2. A system for enlarging thediameter of a vein in a human subject, said vein having a lumen, thesystem comprising:. (a) a catheter configured for injecting apharmaceutical composition directly into a selected segment of the wallof the vein; and (b) a pharmaceutical composition suitable foradministration to the human subject comprising an elastase, wherein saidelastase is present in a dose sufficient to enlarge the diameter of thevein and the diameter of the lumen of the vein when administered via thecatheter to the wall of the vein in the human subject.
 3. The system ofclaim 1 or 2, wherein the catheter comprises a channel for delivery ofsaid pharmaceutical composition.
 4. The system of claim 3, wherein adelivery device containing the pharmaceutical composition is connectedto the catheter for delivering the pharmaceutical composition to theartery or vein via the channel.
 5. The system of claim 4, wherein thedelivery device is a syringe.
 6. The system of claim 1 or 2, which isconfigured for delivering the pharmaceutical composition at low pressureinto the wall of the artery or vein.
 7. The system of claim 1 or 2,wherein the catheter comprises at least one injector port configured forentering the wall of the artery or vein and injecting the pharmaceuticalcomposition directly into the wall of the artery or vein.
 8. The systemof claim 7, wherein said at least one injector port is mounted on thesurface of one or more inflatable balloons.
 9. The system of claim 1 or2, wherein the elastase is a pancreatic elastase.
 10. The system ofclaim 1 or 2, wherein the pharmaceutical composition does not comprise acollagenase.
 11. The system of claim 1 or 2, wherein the cathetercomprises a plurality of injector ports configured for injecting thepharmaceutical composition directly into the wall of the artery or vein12. The system of claim 11, wherein said injector ports can be extendedso as to enter the wall of the artery or vein.
 13. . The system of claim11, wherein said injector ports are mounted on the surface of one ormore inflatable balloons.
 14. The system of claim 1 or 2, wherein thecatheter comprises a reservoir containing the elastase composition.